This invention relates to devices, kits and methods for fluid processing of biological material, particularly processing biological material with controlled and minimal fluid quantities. The devices, kits and methods of the invention are particularly useful for hybridization of biomolecules, which may be bound to a substrate.
High-throughput processing of biological material using fluids containing biomolecules is widely used in several fields. The processing of biological material surface mounted on planar substrates such as slides is known and used in various procedures. For example, biotechnology, biochemistry, molecular biology, molecular genetics, cytogenetics, cell biology, pharmacology, and immunology, are examples of fields in which such procedures have been used for analytical and diagnostic purposes. In a typical procedure, a slide containing immobilized biological material is contacted with a variety of fluids, and usually involves a number of processing steps. It is essential that the processing steps are carried out properly to obtain repeatable and comparable results.
Two examples of such procedures include hybridization of slide-bound nucleic acid probes with labeled targets that incorporate complementary nucleotide sequences and staining of biological material. In a procedure involving the staining of slide mounted specimens of biological materials, the prepared slides may be dipped successively into a series of small vessels or jars, each about 0.2 to about 2 liters in volumetric capacity, and each containing a particular liquid treating composition, such as washing agents, buffers, dehydrating agents, dyes and other solutions. Dye materials may be used to highlight different cells, structures of cells, intracellular structures, and cellular products. Resulting dyed slides may be washed and dried, possibly stored, and then microscopically examined. Selective staining procedures using specific antibody or gene probes have been developed and are advantageously highly specific and sensitive. However, such procedures require many successive steps to be carried out.
Another example of a procedure that utilizes slide-mounted biological materials is hybridization of biomolecules. Screening of biomolecules such as nucleic acids, protein sequences of amino acids, carbohydrates, lipids and living cells containing biomolecules provides information about changes in physiological, biochemical and molecular interactions of biological samples at the cellular and/or subcellular level. Techniques such as hybridization utilize an analyte and a complementary binding entity that form a bound pair of the analyte and the binding entity. Such hybridization can be performed in solution, or alternatively, the analyte can be immobilized on a support such as a glass slide and contacted with a solution containing a binding entity. Typically a pattern or an array of different analytes (usually called probes) are immobilized on a glass slide, and a solution containing a binding entity (usually called the target) contacts the array. Unbound binding entities can be washed from the slide, and various types of analytical technique involving, for example, phosphorescence, fluorescence, and radioactivity, can be performed to determine which specific sites or probes were bound to the target or targets.
In a more specific example, the high throughput screening of nucleic acids is typically performed by attaching base pairs of nucleic acid sequences in an array of locations on a glass plate or slide. Each spot location provides an address for later reference to each spot of nucleic acid. Hybridization techniques utilize markers such as radioactive or fluorescent compounds to label particular nucleic acid sequences that are complementary to the nucleic acid sequences on the glass slide. Signal measurement equipment is then utilized to measure each address on the array to determine if the labeled sequences have attached to the complementary sequence on the glass slide. The resulting slide is examined using an evaluation procedure such as, for example, microscopy, autoradiography, fluorescence measurement, photon emission, or the like. A single hybridization procedure may involve as many as thirty or more controlled step sequences.
Since multi-step processing of slide mounted biological materials typically involves numerous slides and a variety of process liquids and steps, it may be difficult to control the identical treatment of all slides. An additional concern in such processes is that some processing liquids, such as liquids containing nucleotides and other biomolecules, are very costly and must be used in small volumes.
Hybridization reactions are usually carried out in fluid-containing structures such as wells, bottles and other structures designed to contain the target-containing hybridization solution and a substrate containing an array of biomolecular probes. Conventional hybridization chamber devices, particularly hybridization chambers used with microarrays printed on a glass or plastic slide have several shortcomings.
For example, referring to FIGS. 1-7, which shows a typical and commercially available hybridization chamber assembly that includes three layers and a glass slide 10, which is shown in FIG. 1. The glass slide is provided with an array of biomolecules 12 arranged thereon, or an array of biomolecules may be printed on the surface of the glass slide 10. An example of a suitable slide for printing microarrays of biomolecules such as DNA is a CMT-GAPS(trademark) coated slide available from the assignee of the present invention. Referring now to FIG. 3, a frame 14 for a commercially available hybridization chamber frame typically includes three layers. The first layer is a cover sheet 16, which may be made from glass or plastic and may include an inlet opening 18 and an outlet opening 20. The frame includes a second frame layer 22, which typically includes an adhesive backing and is attached to a disposable backing sheet 24.
According to known hybridization procedures, the slide 10 containing an array of biomolecules 12 immobilized on the surface of the slide is typically washed separately before attachment of the hybridization chamber frame. Referring to FIG. 4, after the slide 10 has been washed and dried, the adhesive backing sheet 24 is peeled from the frame layer 22 and the frame layer 22 and cover sheet 16 are positioned over the microarray of biomolecules 12 on the slide 10. Referring to FIG. 5, the frame layer 22 and cover sheet 10 are placed over the microarray of biomolecules 12 to surround the biomolecules and form a well structure 26 with the slide 10.
As shown in FIG. 6, a mixture of hybridization solution containing target molecules is injected in the inlet opening 18 with a pipette 28 or other suitable fluid injection device. Excess fluid may flow out of the outlet opening 20. After injection of the fluid, the hybridization chamber including the frame layer 22, the cover sheet 16 and the slide 10 are left for a time and under conditions to allow hybridization of the target molecules in the hybridization solution and probe biomolecules 12 on the slide 10. Referring now to FIG. 7, the frame layer 22 and the cover sheet 16 are removed from the slide 10, and additional processing steps such as post hybridization washing of the slide 10 containing the biomolecules are performed. Thereafter, the slide 10 is scanned and analyzed.
As evidenced by the above discussion, the delivery and removal of fluids to a hybridization chamber as shown in FIGS. 1-7 is not designed for rapid processing of slide-mounted material. It would be advantageous to provide a chamber frame having connectors that could be readily attached to standard laboratory tubing to facilitate the supply and removal of fluids from the well. Such a chamber would facilitate processing such slide-mounted material in a replicable manner. It would also be useful if the delivery and removal of fluids from such chambers and washing of the slide containing biological material could be automated and did not require laboratory personnel to handle the chambers to perform each step. Still further, there is a need in the art for devices and methods that utilize minimal amounts of processing fluids and agents particularly with regard to hybridization solutions containing target biomolecules.
Accordingly, the present invention generally provides methods, devices and kits for performing biological experiments using fluids. The devices of the invention include a generally planar substrate including a specimen area containing at least one biomolecule. An example of such a planar substrate is a slide containing a microarray of biomolecules. The devices of the present invention further include a frame surrounding at least a portion of the specimen area. The frame defines walls of a well for holding a fluid solution when the frame is in contact with the substrate and the frame including a connector adapted to connect to tubing for supplying fluids to the well.
The devices of the present invention are particularly suitable for use as biomolecular hybridization chambers, such as the type used in hybridization of nucleic acids. According to one aspect of the invention, the connectors are integrally formed with the frame. According to another aspect of the invention, the connectors can be any suitable type for connection with fluid supply tubing. Examples of such connectors include, but are not limited to male luer fittings, female luer fittings, flanged fittings, flangeless fittings, threaded fittings and barbed fittings.
According to another aspect of the invention, the frame includes at least two connectors adapted to connect to tubing for supplying fluids to the well. In this aspect, one of the least two fittings is connected to supply tubing for supplying reagents to the well and one of the at least two fittings is connected to waste tubing for removing fluids from the well.
In another aspect of the invention, the frame is manufactured from an elastomeric material that forms a fluid tight seal with the generally planar substrate. Preferably, the elastomeric material is silicone rubber, poly(dimethylsiloxane) (PDMS), and combinations thereof. PDMS is a particularly preferred material. It is preferred that a material is used so that the frame can be fabricated in relatively small dimensions to provide a frame dimensioned so that the depth of the well is less than 100 microns.
Another aspect of the invention involves a kit for performing experiments with biological materials using fluids including the generally planar substrate and the frame including connectors. Such kits can be utilized to carry out hybridization chamber assays. In this aspect, the kit may further include a slide for printing a high density microarray of biomolecules, or alternatively, a pre-printed DNA micro array.
In another aspect of the invention, a method of performing a hybridization assay is provided. The method includes the steps of providing a generally planar substrate including a specimen area containing at least one biomolecule and a frame including a connector adapted to connect to tubing for supplying fluids to the well. The method further includes the step of placing the frame in contact with the substrate so that the frame surrounds at least a portion of the specimen area and the frame and the substrate define a well for holding a solution.
Another aspect of the invention involves providing a frame including at least two connectors adapted to be connected to tubing. In this aspect, supply tubing may be connected to one of the connectors to supply fluids to the well and waste tubing may be connected to one of the connectors to remove fluids from the well. A further aspect of the invention involves supplying wash fluid to the well through the supply tubing and removing the wash fluid from the well through the waste tubing and supplying hybridization fluid to the well through the supply line. According to this aspect, hybridization material from the well can be removed through the waste tubing.
The invention provides a device, a kit and a method for performing biological experiments. The device allows such experiments to be performed with control over the amount of fluid utilized during such experiments and minimizes the waste of such fluids. The device facilitates the delivery of fluids to a specimen using standard tubing that connects to the connectors included in the hybridization frame. In addition, the connectors associated with frame, which can be connected to standard laboratory tubing, enables faster throughput of samples, which in turn facilitates high throughput screening of biomolecules.
Another advantage of the present invention is that the delivery of hybridization fluid and washing of microarray slides can be efficiently performed without having to remove the hybridization chamber frame. The hybridization solutions and wash fluids can be conveniently delivered via tubing through the connectors included with the hybridization frame. The method requires less laboratory worker skill and handling of reagents, which will lower the cost of laboratory processing and minimize the chance of human error in mishandling samples or possibly using the wrong hybridization solution during testing. Additional advantages of the invention will be set forth in the following detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and are intended to provide further explanation of the invention as claimed.